Study On The Protective Effect And Mechanism Of Qingkailing On The Blood-brain Barrier

ObjectiveBy observing the protective effect of Qingkailing on Ischemia-perfusion injury(I/R)in mice,and the influence on the blood-brain barrier-related pathway,and the mechanism of anticerebral ischemia is discussed from the perspective of the intervention of blood-brain barrier.Methods1.Male C57bl/6 mice were randomly divided into four groups: sham group,model ischemic(Ischemia)group,Qingkailing(QKL)group(3ml·kg-1),edaravone(EDA)group(3ml·kg-1),The neurological deficit was assessed by the neurological function score scoring method by preparing the MCAO mouse model.Determination of cerebral infarction volume by TTC staining;HE staining to observe the morphological changes of glial cells;Nissl staining method to observe the situation of Nissl bodies.Furthermore,the protective effect of Qingkailing on mouse I/R was explored.2.Male C57bl/6 mice were randomly divided into four groups: sham group,model ischemic(Ischemia)group,Qingkailing(QKL)group(3ml·kg-1),edaravone(EDA)group(3ml·kg-1).The MCAO mouse model was prepared,and the microvascular structure was observed dynamically by two-photon microscopy.The blood-brain barrier permeability was observed by Evans blue test.The ultrastructure of the blood-brain barrier was observed by transmission electron microscopy.Western-blot was used to immunize.The histochemical method was used to detect the expression of HIF-1?,MMP-9,occludin,ZO-1,and claudin-5 in the brain tissue of ischemic hemisphere.From the perspective of the intervention of the bloodbrain barrier,the mechanism of action of Qingkailing active ingredients against cerebral ischemia was revealed.ResultsNeurological function scoreThe sham operation group had no neurological impairment behavior,and the neurological deficit score was scored 24 hours after cerebral ischemia-reperfusion(I/R).The results showed that the score of the model ischemic group(Ischemia group)was significantly increased at 24 hours compared with the sham operation group(Sham group).However,after treatment with Qingkailing(QKL)or edaravone(EDA),the mice in the QKL group and the EDA group had significantly lower scores than the mice in the ischemic group(P<0.001).These results indicate that QKL improves neurological deficits in a manner that indicates a reduction in the volume of cerebral infarction.TTC stainingThe Sham group showed a uniform distribution of red by TTC staining,while the Ischemia group showed significant ischemic lesions.Compared with the model group,QKL-treated mice significantly reduced cerebral infarction volume after 24 hours of I/R and had the same efficacy as EDA(P < 0.001).The blood-brain barrier is part of the neurovascular unit,so the next experiment focuses on the protective effects of QKL on brain neuronal cells and glial cells.HE stainingThere was no obvious pathological change in the brain tissue of Sham group.The cell morphology was normal.The cells and glial cells were arranged neatly and orderly,the nucleus was large,the chromatin was evenly distributed,and the nucleolus was clear.In the Ischemia group,a large infarct area was seen,which was liquefied necrosis(softening lesion).Large pieces of nerve cells disappeared,and a large amount of glial cells proliferated in the necrotic area.The necrotic margin(semi-dark band)was narrow,and a large amount of neutrophil infiltration was observed.The nerve cells are condensed into triangles and the nucleus is darkly stained.The number of neurons in the QKL group and the EDA group was slightly reduced,mildly degenerated,more red neurons,and the proliferation of microglia was not obvious.Nissl stainingThe glial cells in the Sham group are arranged neatly and orderly,no edema is seen,and the cell body and nucleolus are clearly visible,and the Nissl body is more common.In the Ischemia group,the Nissl bodies were significantly reduced,and some of the nerve cells were condensed and deeply stained.After administration of QKL and EDA,the infarct size was reduced,the number of intact neurons was large,and the range of degeneration and necrosis was relatively small.The statistical results showed that the number of Nissl-stained apoptotic neurons in the EDA and QKL groups was significantly lower than that in the Ischemia group(P < 0.001).Cerebral cortex microvascular structureHere we show the destruction of microvascular structures in the blood-brain barrier dysfunction after cerebral ischemia.The Sham group showed vascular patency,while the Ischemia group showed nodular contraction.The QKL and EDA groups significantly reduced the cerebral vasculature after cerebral ischemia.After 24 hours of cerebral ischemia,the vascular morphology changed more severely after 4 hours.Blood-Brain Barrier permeabilityOur quantitative results showed a significant increase in EB levels in Ischemia compared with the Sham group 24 hours after cerebral ischemia-reperfusion,indicating a discontinuation of BBB after cerebral ischemia-reperfusion.However,after treatment with QKL or EDA for 24 hours,the EB content of the mice in the ipsilateral hemisphere was significantly reduced compared to the ischemia group.The results of this method indicate that QXL can attenuate the destruction of BBB and protect BBB in mice with cerebral ischemia-reperfusion.Tight junction ultrastructureThree mice from each treatment group were randomly assigned for tight junction ultrastructural studies using transmission electron microscopy.The tight junction between two adjacent endothelial cells of the Sham group appears as a typical thickened dark line of the cell membrane alone,representing the normal expression of connexin.In contrast,the tight junctions of the Ischemia group were significantly impaired and the connexin layer was thinner.Endothelial cells also become edematous and show cytoplasmic vacuolation and mitochondrial degeneration.Abnormal ultrastructure of tight junctions in QKL group and EDA group was significantly weakened,indicating that QKL / EDA treatment attenuated tight junctional damage caused by cerebral ischemia-reperfusion.Expression of blood-brain barrier-related antibodiesIn the I/R model,I-R was able to induce HIF-1? and activate HIF-1?/MMP-9 pathway by western-blot and immunohistochemical assays,on the one hand after cerebral ischemia and reperfusion.The expressed HIF-1? can promote the expression of MMP-9.On the other hand,the highly expressed MMP-9 can promote the breakdown of the tight junction proteins occludin,ZO-1,and claudin-5.Qingkailing(QKL)and the positive drug edaravone(EDA)can reduce the expression of HIF-1? and MMP-9,increase the expression of occludin,ZO-1,and claudin-5,and promote the structure of the blood-brain barrier.The recovery of function reduces the damage of the blood-brain barrier.Conclusion1.Qingkailing can reduce the volume of cerebral infarction in mice with cerebral ischemia-reperfusion,improve the neurological function score,and improve the pathological damage of brain tissue,suggesting that Qingkailing has protective effect on I/R.2.After 24 hours of cerebral ischemia-reperfusion injury in mice,Qingkailing intervention reduced the microvascular morphology and permeability of the blood-brain barrier after cerebral ischemia,and improved the tightly connected ultrastructure.It shows that Qingkailing can reduce the damage of the blood-brain barrier and protect the blood-brain barrier.3.After 24 hours of cerebral ischemia-reperfusion injury,the expression of HIF-1? was significantly increased,and the HIF-1?/MMP-9 pathway was activated.After administration,the expression of HIF-1? and MMP-9 was decreased,and the expression of tight junction proteins occludin,ZO-1,and claudin-5 was increased,suggesting that Qingkailing promotes the recovery of blood-brain barrier structure and function.